ABSTRACT This proposal applies a microfluidic oxygen gradient with multimodal stimulations and detections to investigate a possible link between hypoxia and free fatty acid (FFA) exposures in beta cell impairments and apoptosis. Distinct from the autoimmune pathology in type I diabetes, type II diabetes development relating to beta cell dysfunction is not well understood, including numerous pathological mechanisms and risk factors. Many risk factors coexist in obesity and could possibly interact synergistically. For example, elevated FFA in obesity can lead to insulin deficiency, although only a fraction of people with high FFA develop diabetes. On the other hand, hypoxia can also suppress insulin secretion, but no clear mechanism leading to the development of diabetes has been discovered. Intriguingly, recent studies suggest that both FFA and hypoxia exacerbate beta cell ER stress via the unfolded protein response (UPR) pathways, possible through reactive oxygen species (ROS). However, neither factor was sufficient to induce beta cell apoptosis alone. This suggests that a synergistic effect might exist, as hypoxia also affects FFA levels in vivo. Despite suggestive evidences of this fatty acid-hypoxia link, however, its potential role in islet pathophysiology has not been demonstrated. One reason is the lack of proper techniques for controlling hypoxia at the microscale level of beta cells. Standard hypoxic chambers cannot generate a gradient to test multiple exposures, nor can they provide in situ microscopy of beta functions. Proposed here is a multimodal microfluidic device with integrated oxygen gradient for hypoxia and FFA exposures. It incorporates an open-top design to allow easy access for normal cell culturing and assay reagent exchange. Furthermore, a novel hydrogel sensor film is layered directly beneath the cells to provide in situ spatial insulin detections, in addition to spatial monitoring of Ca2+ and ROS via microscopy. Aim 1 applies this gradient to probe multiple hypoxia and FFA concentrations individually in the impairment of beta function and viability. Aim 2 will test the combinatory effects of both factors at or below their transition concentrations in a stimulation matrix. The key technology is using microfluidics to discretely control each of the factors, enabling studies of their interactions in well-controlled beta cell cultures, leading to a more deterministic results. By proving the hypothesis of FFA-hypoxia synergy in inducing beta cell impairment and apoptosis, a potential mechanism can be proposed towards the complicated development of type II diabetes. The results obtained from these feasibility experiments will provide target parameters to be tested in a larger high-throughput microfluidic array.